Using CRISPR/Cas9 technology, researchers have devised a method to deliver a CAR gene to a specific locus, TRAC, in T cells. This targeted approach yielded therapeutic cells that were more potent even at low doses; in a mouse model of acute lymphoblastic leukemia, they outperformed CAR T cells created with a randomly integrating retroviral vector.
In a preclinical study merging gene editing with chimeric antigen receptor (CAR) T-cell therapy, researchers at Memorial Sloan Kettering Cancer Center (MSKCC) in New York, NY, have devised a method to improve CAR gene delivery, thereby considerably enhancing the potency of these modified T cells.
Until now, retroviral vectors have been the delivery method of choice, but “this means the gene can integrate anywhere in recipient cells, leading to substantial differences in CAR expression levels,” says senior author Michel Sadelain, MD, PhD, director of MSKCC's Center for Cell Engineering. Instead, by harnessing CRISPR/Cas9 technology to strategically place a CD19-recognizing CAR gene in the T-cell receptor α-chain constant (TRAC) locus in human peripheral blood T cells, “we achieved uniform CAR expression across cells from multiple donors,” Sadelain says.
Importantly, these TRAC–CAR T cells outperformed conventionally generated CD19-targeting CAR T cells in a mouse model of acute lymphoblastic leukemia. When the “CAR stress test” was carried out—gradually lowering therapy dosage to assess the cells' potency—TRAC–CAR T cells induced greater reductions in tumor burden and prolonged the median survival at every dose.
“We tried inserting our CAR gene at multiple other sites, but compared with TRAC, the T cells were simply not as effective,” Sadelain observes. His team found that when TRAC was used as the point of integration, the level of CAR expression was not only consistent across all cells but just right, he says, to prevent tonic signaling, or continuous CAR activity: a phenomenon that exhausts T cells and accelerates their terminal differentiation. Upon antigen binding, regular CAR turnover—internalization followed by reexpression on the cell surface, for further antitumor activity—also proceeded at an optimal pace.
“With conventional CAR T cells, or when using other loci besides TRAC, we saw that sometimes internalization didn't fully happen, or the CAR rebounded too quickly,” Sadelain explains. “Essentially, CAR regulation in T cells is subtle and exquisite; there's a whole range of nuances we're only just uncovering.”
He points out, too, that because using TRAC requires first disabling the native T-cell receptor in the same locus, “we can be more confident that these modified cells may be safely used in patients with a preexisting autoimmune condition. Right now, such patients are often excluded from CAR T-cell therapy trials.” This approach should also allow for an allogeneic, “off-the-shelf” therapy that doesn't have to be derived from a patient's own T cells.
In an accompanying commentary, Marcela Maus, MD, PhD, director of cellular immunotherapy at Massachusetts General Hospital Cancer Center in Boston, noted that targeted CAR gene integration “might prove safer than random integration, which carries the potential risk of generating a harmful mutation.” Given the potency of TRAC–CAR T cells even at low doses, this strategy should enable faster, cheaper manufacturing, she added. However, whether it improves the toxicity profile of CAR T cells—cytokine release syndrome poses a significant clinical challenge—is still undetermined.
Having successfully scaled up their technique, Sadelain and his team will soon seek the FDA's approval to evaluate TRAC–CAR T cells in humans. Overall, given the field's rapid progress, he thinks “the first approved CAR T-cell therapy will probably look obsolete quite soon afterwards, at least in the research world. Better and better iterations are being developed.” –Alissa Poh